Use of De Novo Designed Peptides for the Study of Metalloproteins and Enzymes

Dieckmann, Gregg R.; Heilman, Sandy; McRorie, Donald K.; DeGrado, William F.; Pecoraro, Vincent L.

doi:10.1007/978-94-011-0255-1_21

Cited by 3 publications

(3 citation statements)

References 8 publications

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“…The typical sequence that we utilize is based on the heptad repeat LKALEEK [112]. Notice that the a and d positions contain the bulky hydrophobe leucine.…”

Section: Lead Chemistry With Designed Proteinsmentioning

confidence: 99%

10. Lead(II) Binding in Natural and Artificial Proteins

Cangelosi¹,

Ruckthong²,

Pecoraro³

2017

Lead – Its Effects on Environment and Health

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This article describes recent attempts to understand the biological chemistry of lead using a synthetic biology approach. Lead binds to a variety of different biomolecules ranging from enzymes and regulatory and signaling proteins to bone matrix. We have focused on the interactions of this element in thiolate-rich sites that are found in metalloregulatory proteins such as Pbr, Znt, and CadC and in enzymes such as δ-aminolevulinic acid dehydratase (ALAD). In these proteins, Pb(II) is often found as a homoleptic and hemidirectic Pb(II)(SR)3− complex. Using first principles of biophysics, we have developed relatively short peptides that can associate into three-stranded coiled coils (3SCCs), in which a cysteine group is incorporated into the hydrophobic core to generate a (cysteine)3 binding site. We describe how lead may be sequestered into these sites, the characteristic spectral features may be observed for such systems and we provide crystallographic insight on metal binding. The Pb(II)(SR)3− that is revealed within these α-helical assemblies forms a trigonal pyramidal structure (having an endo orientation) with distinct conformations than are also found in natural proteins (having an exo conformation). This structural insight, combined with 207Pb NMR spectroscopy, suggests that while Pb(II) prefers hemidirected Pb(II)(SR)3− scaffolds regardless of the protein fold, the way this is achieved within α-helical systems is different than in β-sheet or loop regions of proteins. These interactions between metal coordination preference and protein structural preference undoubtedly are exploited in natural systems to allow for protein conformation changes that define function. Thus, using a design approach that separates the numerous factors that lead to stable natural proteins allows us to extract fundamental concepts on how metals behave in biological systems.

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“…The typical sequence that we utilize is based on the heptad repeat LKALEEK [112]. Notice that the a and d positions contain the bulky hydrophobe leucine.…”

Section: Lead Chemistry With Designed Proteinsmentioning

confidence: 99%

10. Lead(II) Binding in Natural and Artificial Proteins

Cangelosi¹,

Ruckthong²,

Pecoraro³

2017

Lead – Its Effects on Environment and Health

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“…The sequence chosen to achieve our original goal is TRI L9CL23H, where His at position 23 is intended for formation of a ZnHis 3 X site. In addition to the envisioned catalytic zinc site, we engineered a stabilizing structural site (HgS 3 , where S represents a thiolate ligand) utilizing principles for heavy metal binding defined by earlier work in the group. ,− Having a structural site was initially desirable in part to support substitution of the bulky, potentially destabilizing, His residues into the interior of the coiled coil. X-ray crystallographic analysis does demonstrate some fraying of the coiled coil below the His site (Figure ), and circular dichroism (CD) studies indicate somewhat lower α-helical content for His-containing peptides (∼70–80%) than for those that contain single Cys substitutions (>90%).…”

Section: From Zinc-binding Proteins To Hydrolytic Metalloenzymesmentioning

confidence: 99%

Designing Hydrolytic Zinc Metalloenzymes

2014

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Zinc is an essential element required for the function of more than 300 enzymes spanning all classes. Despite years of dedicated study, questions regarding the connections between primary and secondary metal ligands and protein structure and function remain unanswered, despite numerous mechanistic, structural, biochemical, and synthetic model studies. Protein design is a powerful strategy for reproducing native metal sites that may be applied to answering some of these questions and subsequently generating novel zinc enzymes. From examination of the earliest design studies introducing simple Zn(II)-binding sites into de novo and natural protein scaffolds to current studies involving the preparation of efficient hydrolytic zinc sites, it is increasingly likely that protein design will achieve reaction rates previously thought possible only for native enzymes. This Current Topic will review the design and redesign of Zn(II)-binding sites in de novo-designed proteins and native protein scaffolds toward the preparation of catalytic hydrolytic sites. After discussing the preparation of Zn(II)-binding sites in various scaffolds, we will describe relevant examples for reengineering existing zinc sites to generate new or altered catalytic activities. Then, we will describe our work on the preparation of a de novo-designed hydrolytic zinc site in detail and present comparisons to related designed zinc sites. Collectively, these studies demonstrate the significant progress being made toward building zinc metalloenzymes from the bottom up.

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“…25 These peptides provide us with simpler constructs for understanding more complicated native proteins.. The successes using this strategy include the first spectroscopic and structural models 24,26–28 for mercury(II) binding to MerR, arsenic(III) complexation 29,30 by ArsR and trigonal 31 cadmium(II) for CmtR. We have also demonstrated how to control the coordination geometry of metal ions such as cadmium(II) at the peptide interior.…”

Section: Introductionmentioning

confidence: 98%

Experimental and Theoretical Evaluation of Multisite Cadmium(II) Exchange in Designed Three-Stranded Coiled-Coil Peptides

Chakraborty¹,

Iranzo

Zuiderweg³

et al. 2012

J. Am. Chem. Soc.

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An important factor that defines the toxicity of elements such as cadmium(II), mercury(II), and lead(II) with biological macromolecules is metal ion exchange dynamics. Intriguingly, little is known about the fundamental rates and mechanisms of metal ion exchange into proteins, especially helical bundles. Herein, we investigate the exchange kinetics of cadmium(II) using de novo designed three-stranded coiled coil peptides that contain metal complexing cysteine thiolates as a model for the incorporation of this ion into trimeric, parallel helical bundles. Peptides were designed containing both single cadmium(II) binding site, GrandL12AL16C [Grand=AcG-(LKALEEK)5-GNH2], GrandL26AL30C, and GrandL26AE28QL30C, as well as GrandL12AL16CL26AL30C with two cadmium(II) binding sites. The binding of cadmium(II) to any of these sites is of high affinity (KA > 3×107 M−1). Using 113Cd NMR spectroscopy, cadmium(II) binding to these designed peptides was monitored. While the cadmium(II) binding is in extreme slow exchange without showing any chemical shift changes, incremental line broadening for the bound 113cadmium(II) signal is observed when excess 113cadmium(II) is titrated into the peptides. Most dramatically, for one site, L26AL30C, all 113cadmium(II) NMR signals disappear once a 1.7:1 ratio of cadmium(II)/(peptide)3 is reached. The observed processes are not compatible with simple “free-bound” two-site exchange kinetics at any time regime. The experimental results can, however, be simulated in detail with a multi-site binding model, which features additional cadmium(II) binding site(s) which, once occupied, perturb the primary binding site. This model is expanded into differential equations for five-site NMR chemical exchange. The numerical integration of these equations exhibits progressive loss of the primary site NMR signal without a chemical shift change and with limited line broadening, in good agreement with the observed experimental data. The mathematical model is interpreted in molecular terms as representing binding of excess cadmium(II) to surface Glu residues located at the helical interfaces. In the absence of cadmium(II), the Glu residues stabilize the three-helical structure though salt bridge interactions with surface Lys residues. We hypothesize that cadmium(II) interferes with these surface ion pairs, destabilizing the helical structure, and perturbing the primary cadmium(II) binding site. This hypothesis is supported by the observation that the cadmium(II)-excess line broadening is attenuated in GrandL26AE28QL30C where a surface Glu(28), close to the metal binding site, was changed to Gln. The external binding site may function as an entry pathway for cadmium(II) to find its internal binding site following a molecular rearrangement which may serve as a basis for our understanding of metal complexation, transport and exchange in complex native systems containing α-helical bundles.

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Use of De Novo Designed Peptides for the Study of Metalloproteins and Enzymes

Cited by 3 publications

References 8 publications

10. Lead(II) Binding in Natural and Artificial Proteins

10. Lead(II) Binding in Natural and Artificial Proteins

Designing Hydrolytic Zinc Metalloenzymes

Experimental and Theoretical Evaluation of Multisite Cadmium(II) Exchange in Designed Three-Stranded Coiled-Coil Peptides

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